Intracellular calcium concentration ([Ca2+]i) is an important regulator of keratinocyte proliferation and differentiation. Hence, changes in [Ca2+]i play an important role in keratinocyte functions involved with tissue differentiation, wound healing, cancer development and bacterial invasion. The Principal Investigator found recently that nuclear [Ca2+] ([Ca2+]n) changes occur during bacterial invasion of keratinocytes. These [Ca2+]n changes were also dependent on Ca2+ influx. Moreover, the magnitude of the store depletion induced capacitative current was found to depend on the differentiation status of the keratinocyte. Since [Ca2+]n changes are important regulators of gene expression in all cells, such changes are likely to play an important role in the host response to bacterial invasion. However, before this invasion process can be understood, the mechanisms by which [Ca2+]n is regulated must be elucidated. In addition, it is necessary to elucidate why the capacitative current varies with keratinocyte differentiation state. The present proposal initiates efforts to understand these [Ca2+] regulatory mechanisms. Hence, this proposal has the following Specific Aims: Specific Aim 1 proposes to determine how intracellular Ca2+ stores affect nuclear [Ca2+]. Specific Aim 2 proposes to characterize the Ca2+ permeable channels activated by Ca2+ store depletion, and to determine whether the resulting Ca2+ influx alters nuclear [Ca2+]. Specific Aim 3 will demonstrate that the nuclear Ca2+ signaling system is altered in Ca2+-activated, differentiated keratinocytes as compared to no differentiated keratinocytes. [Ca2+]i will be measured with wide-field, deconvolution microscopy using fura-2; Ca2+ channel properties with whole-cell and single channel, patch clamp techniques. Keratinocyte differentiation will be induced by exposure to Ca2+ in culture, and monitored by induction of involucrin and cytokeratin 13. Completion of the proposed Specific Aims will establish the characteristics of the capacitatively activated Ca current and the Ca stores that affect [Ca2+]n in differentiated and undifferentiated keratinocytes, and their relationships to the [Ca2+]n changes that can affect gene expression.